Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/02—Bacterial antigens
  • A61K39/07—Bacillus
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/66—Microorganisms or materials therefrom
  • A61K35/74—Bacteria
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/66—Microorganisms or materials therefrom
  • A61K35/74—Bacteria
  • A61K35/741—Probiotics
  • A61K35/742—Spore-forming bacteria, e.g. Bacillus coagulans, Bacillus subtilis, clostridium or Lactobacillus sporogenes
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/14—Antivirals for RNA viruses
  • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/20—Antivirals for DNA viruses
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/04—Immunostimulants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
  • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
  • A61K2039/521—Bacterial cells; Fungal cells; Protozoal cells inactivated (killed)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
  • A61K2039/541—Mucosal route
  • A61K2039/542—Mucosal route oral/gastrointestinal
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
  • A61K2039/572—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 cytotoxic response

Definitions

• the invention relates to the use of viable and no n- viable bacteria to boost the immune system.
• the gastrointestinal microflora plays a number of vital roles in maintaining gastrointestinal tract function and overall physiological health.
• the growth and metabolism of the many individual bacterial species inhabiting the gastrointestinal tract depend primarily upon the substrates available to them, most of which are derived from the diet. See, e.g., Gibson G.R. et al, 1995 Gastroenterology 106: 975-982; Christl, S.U. et al, 1992 Gut 33: 1234-1238.
• Probiotic organisms are non-pathogenic, non-toxigenic, retain viability during storage, and typically survive passage through the stomach and small intestine. Since probiotics do not generally permanently colonize the host, they need to be ingested regularly for any health promoting properties to persist.
• the invention describes the use of lactic acid-producing bacteria or non-viable fragments or products thereof to boost the immune system.
• the administration of Bacillus coagulans, purified cell wall components of Bacillus coagulans, or culture supernatants of Bacillus coagulans increases the immune system's ability to fight pathogens.
• Cell wall components and/or culture supernatants are useful in products where conditions are not optimal for long-term vegetative cell viability, e.g., shelf stable beverages or food compositions.
• inactivated/dead Bacillus coagulans e.g., heat killed Bacillus coagulans is administered to boost the immune system.
• the methods optionally include administration of purified viable Bacillus coagulans vegetative cells and/or spores to boost the immune system.
• a method of enhancing or boosting an immune response to a microbial pathogen is carried out by identifying a subject infected with a microbial pathogen and administering to the subject a composition comprising a viable Bacillus coagulans bacterium, a no n- viable fragment of the bacterium, or a non-viable extracellular product of the bacterium.
• the bacteria, fragments, or products are administered in an amount that enhances the immune response of the subject to the pathogen with which the subject is infected.
• the bacteria, fragments, or products are purified or fractionated from other bacteria or other components of the bacteria (in the case of fragments, e.g., cell wall fragments or secreted products).
• Purified and/or isolated Bacillus coagulans is particularly useful as a probiotic in the methods and compositions described herein.
• purified or “substantially purified” is meant a Bacillus coagulans bacterium, a non-viable fragment of the bacterium, or a no n- viable extracellular product of the bacterium that is substantially free of contaminating microorganisms or other macromolecules, e.g., polysaccharides, nucleic acids, or proteins.
• a purified preparation contains at least 75%, 85%, 95% or 100% of the desired composition and is substantially free of other sub-cellular components such as cytoplasmic organelles.
• a bacterial cell wall fraction is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% cell wall fragments.
• Such a preparation is sustainably free of cytoplasm intracellular organelles and secreted bacterial products.
• the microbial pathogen is a bacterium or virus such as a pathogen that causes a respiratory infection.
• the pathogen comprises an influenza virus such as a human, avian, or swine influenza virus or combination thereof.
• influenza virus such as a human, avian, or swine influenza virus or combination thereof.
• Other viral pathogens include adenovirus.
• compositions of the invention comprise an immune- enhancing amount of a viable Bacillus coagulans bacterium, a non-viable fragment of Bacillus coagulans bacterium, or a nonviable extracellular product of Bacillus coagulans bacterium ⁇ e.g., a supernatant of a Bacillus coagulans bacterium).
• Enhancement of the immune response comprises an increase in cytokine (e.g., interleukin-2 (IL-2), IL-4, IL-6, IL-10, tumor necrosis factor- ⁇ (TNF- ⁇ ), and interferon- ⁇ (IFN- ⁇ ) production or an increase in immune cell migration to an infection site.
• cytokine e.g., interleukin-2 (IL-2), IL-4, IL-6, IL-10, tumor necrosis factor- ⁇ (TNF- ⁇ ), and interferon- ⁇ (IFN- ⁇ ) production or an increase in immune cell migration to an infection site.
• Immune enhancement also includes boosting the immune system by increasing cytokine production, activating the immune surveillance aspect of polymorphonuclear leukocytes (PMN), increasing immune cell chemotaxis, activating NK cells, and/or increasing monocyte phagocytosis.
• PMN polymorphonuclear leukocytes
• the compositions of the invention increase the chemotactic abilities and phagocytic abilities of PMNs.
• the compositions of the invention also increase the expression of CD69 on NK cells.
• an immune-enhancing amount of Bacillus coagulans, Bacillus coagulans supernatant, or Bacillus coagulans cell wall components is about 0.1 mg to about 10 grams, e.g., about 1 mg to about 10 grams, about 10 mg to about 5 grams; about 100 mg to about 1 gram; or about 200 mg to about 1 gram.
• compositions suitable for human ingestion such as a composition comprising a purified cell wall of a Bacillus coagulans bacterium or a composition comprising a dried or lyophilized secreted product or mixture of secreted products of Bacillus coagulans.
• exemplary formulations include a pill, capsule, or suspension.
• Exemplary bacterial species for the compositions and methods described herein include Bacillus coagulans, e.g., Bacillus coagulans hammer, preferably Bacillus coagulans hammer strain Accession No. ATCC 31284, or one or more strains derived from Bacillus coagulans hammer strain Accession No. ATCC 31284 (e.g., ATCC Numbers: GBI-20 (GB-20), ATCC Designation Number PTA-6085; GBI-30 (GB-30/Ganeden BC 30 TM/BC 30 ), ATCC Designation Number PTA-6086; and GBI-40 (GB-40), ATCC Designation Number PTA-6087; see, U.S. Patent No. 6,849,256 to Farmer).
• the Bacillus coagulans comprises GBI-30 (BC 30 ), or any strain of the organism described in U.S. S.N. 11/706,642, hereby incorporated by reference.
• the Bacillus coagulans Hammer strains of the invention are non-pathogenic and generally regarded as safe for use in human nutrition (i.e., GRAS classification) by the U.S. Federal Drug Administration (FDA) and the U.S. Department of Agriculture (USDA), and by those skilled in the art. Furthermore, the Bacillus coagulans Hammer strains of the invention germinate at or below human body temperature, rendering them useful as probiotics. Many Bacillus coagulans strains outside the Hammer group have mostly industrial applications, little or no nutritional benefit, and environmental contaminants that have not been evaluated for safety. Moreover, many other non-Hammer strains of Bacillus coagulans grow optimally at temperatures that exceed human body temperature and, thus, do not germinate efficiently in the human body.
• Figure l is a line graph demonstrating the percent inhibition of spontaneous reactive oxygen species (ROS) formation after polymorphonuclear leukocyte (PMN) exposure to either Bacillus coagulans supernatant (BCl) or Bacillus coagulans cell wall components (BC2), compared to baseline results.
• ROS spontaneous reactive oxygen species
• Figure 2 is a line graph illustrating the percent inhibition of H 2 ⁇ 2 -induced ROS formation compared to baseline results.
• Figure 3 is a schematic representation of how PMN migration begins in the blood stream and moves into the tissue via transwell migration plates.
• Figure 4 is a line graph demonstrating random migration showing the migratory patterns of PMN's treated with either Bacillus coagulans supernatant (BCl) or Bacillus coagulans cell wall components (BC2).
• BCl Bacillus coagulans supernatant
• BC2 Bacillus coagulans cell wall components
• Figure 5 is a line graph illustrating bacterial peptide formyl-Met-Leu-Phe (f-MLP)- directed migration showing the migratory patterns of PMS 's treated with either Bacillus coagulans supernatant (BCl) or Bacillus coagulans cell wall components (BC2).
• BCl Bacillus coagulans supernatant
• BC2 Bacillus coagulans cell wall components
• Figure 6A is a line graph demonstrating interleukin-8 (IL-8)-directed migration showing the migratory patterns of PMN's treated with either Bacillus coagulans supernatant (BCl) or Bacillus coagulans cell wall components (BC2).
• Figure 6B is a line graph depicting IL-8 directed migration of Bacillus coagulans supernatant (BCl) and Bacillus coagulans cell wall components (BC2)-treated PMN cells.
• Figure 7A is a line graph illustrating leukotriene B4 (LTB4)-directed migration showing the migratory patterns of PMN's treated with either Bacillus coagulans supernatant (BCl) or Bacillus coagulans cell wall components (BC2).
• Figures 7B-7C are a series of bar charts illustrating LTB4-directed migration.
• Figure 8 is a bar graph demonstrating synergistic random migration showing the migratory patterns of PMN's exposed to either Bacillus coagulans supernatant (BCl) ox Bacillus coagulans cell wall components (BC2) acting as a chemoattractant in the bottom chamber of the trans-well migration plate.
• BCl Bacillus coagulans supernatant
• BC2 Bacillus coagulans cell wall components
• Figure 9 is a bar chart illustrating synergistic f-MLP-directed migration showing the migratory patterns of PMN's exposed to either Bacillus coagulans supernatant (BCl) ox Bacillus coagulans cell wall components (BC2) acting as a chemoattractant in the bottom chamber of the trans-well migration plate.
• BCl Bacillus coagulans supernatant
• BC2 Bacillus coagulans cell wall components
• Figure 10 is a bar graph demonstrating the synergistic IL-8-directed migration showing the migratory patterns of PMN's exposed to either Bacillus coagulans supernatant (BCl) or Bacillus coagulans cell wall components (BC2) acting as a chemoattractant in the bottom chamber of the trans-well migration plate.
• BCl Bacillus coagulans supernatant
• BC2 Bacillus coagulans cell wall components
• Figure 11 is a bar chart demonstrating the synergistic LTB4-directed migration showing the migratory patterns of PMN's exposed to either Bacillus coagulans supernatant (BCl) or Bacillus coagulans cell wall components (BC2) acting as a chemoattractant in the bottom chamber of the trans-well migration plate.
• BCl Bacillus coagulans supernatant
• BC2 Bacillus coagulans cell wall components
• Figure 12 is a bar graph illustrating the monocyte phagocytosis as measured by how well the monocyte can ingest green carboxylate fluorspheres.
• Figure 13 is a bar chart demonstrating PMN phagocytosis as measured by how well the monocyte can ingest green carboxylate fluorspheres.
• Figure 14 is a line graph showing cluster of differentiation 69 (CD69) expression of natural killer (NK) cells (analysis generated by measuring the mean fluorescence intensity (MFI) of CD69).
• Figure 15 is a line graph showing CD25 expression of NKT cells (analysis generated by measuring the MFI of CD25).
• Figure 16 is a line graph demonstrating CD 107a expression of NK cells (analysis generated by measuring the MFI of CD 107a).
• Figure 17 is a line graph illustrating the results of lymphocytes that were pre -treated with either Bacillus coagulans supernatant (BCl) or Bacillus coagulans cell wall components (BC2) and then exposed to the mitogen phytohemagglutinin (PHA).
• BCl Bacillus coagulans supernatant
• BC2 Bacillus coagulans cell wall components
• Figure 18 is a line graph demonstrating the results of lymphocytes that were pre-treated with either Bacillus coagulans supernatant (BCl) or Bacillus coagulans cell wall components (BC2) and then exposed to the pokeweed mitogen (PWM).
• BCl Bacillus coagulans supernatant
• BC2 Bacillus coagulans cell wall components
• Figures 19A and B are line graphs showing the cytokine production of lymphocytes pre- treated with Bacillus coagulans supernatant (BCl).
• Figures 2OA and B are line graphs illustrating the cytokine production of lymphocytes pre-treated with Bacillus coagulans cell wall components (BC2).
• Figure 21 is a bar chart showing the results of lymphocytes that were pre-treated with either Bacillus coagulans supernatant (BCl) or Bacillus coagulans cell wall components (BC2), and then exposed to no mitogen, PHA, or PWM. This graph represents relative levels of the cytokine IL-2 present in the supernatant of 5 day lymphocyte cultures. Untreated (UT).
• Figure 22 is a bar graph illustrating the results of lymphocytes that were pre-treated with either Bacillus coagulans supernatant (BCl) or Bacillus coagulans cell wall components (BC2) and then exposed to no mitogen, PHA, or PWM. This graph represents relative levels of the cytokine IL-4 present in the supernatant of 5 day lymphocyte cultures.
• BCl Bacillus coagulans supernatant
• BC2 Bacillus coagulans cell wall components
• Figure 23 is a bar chart demonstrating the results of lymphocytes pre-treated with either Bacillus coagulans supernatant (BCl) or Bacillus coagulans cell wall components (BC2), and then exposed to no mitogen, PHA, or PWM. This graph represents relative levels of the cytokine IL-6 present in the supernatant of 5 day lymphocyte cultures.
• BCl Bacillus coagulans supernatant
• BC2 Bacillus coagulans cell wall components
• Figure 24 is a bar graph illustrating the results of lymphocytes that were pre-treated with either Bacillus coagulans supernatant (BCl) or Bacillus coagulans cell wall components (BC2), and then exposed to no mitogen, PHA, or PWM. This graph represents the relative levels of the cytokine IL-IO present in the supernatant of 5 day lymphocyte cultures.
• BCl Bacillus coagulans supernatant
• BC2 Bacillus coagulans cell wall components
• Figures 25 A and B shows a series of bar charts showing the results of lymphocytes that were pre-treated with either Bacillus coagulans supernatant (BCl) or Bacillus coagulans cell wall components (BC2), and then exposed to no mitogen, PHA, or PWM.
• This graph represents the relative levels of the cytokine TNF- ⁇ present in the supernatant of 5 day lymphocyte cultures.
• Figures 26 A and B show a series of bar charts showing the results of lymphocytes that were pre-treated with either Bacillus coagulans supernatant (BCl) or Bacillus coagulans cell wall components (BC2), and then exposed to no mitogen, PHA, or PWM.
• This graph represents relative levels of the cytokine IFN- ⁇ present in the supernatant of 5 day lymphocyte cultures.
• Figure 27 is a schematic of a cell-based antioxidant protection in erythrocytes (CAP-e) representation of how a natural product gets into the cell.
• a dye is used to express fluorescence representing oxidative stress.
• Figure 28 is a line graph showing CAPe results for Bacillus coagulans supernatant being tested in parallel on fresh and aged cells.
• Figure 29 is a line graph showing CAPe results for Bacillus coagulans cell wall being tested in parallel on fresh and aged cells.
• Figure 3OA is a photomicrograph depicting an untreated PMN cell that has engaged in phagocytosis.
• the green beads are carboxylated fiuorospheres that mimic bacterial particles.
• Figure 3OB is a photomicrograph depicting a PMN cell treated with Bacillus coagulans cell wall components (BC2).
• Figure 31 is an illustration of a typical protein gel electrophoresis method.
• Figure 32 is a photograph depicting the results of a gel electrophoresis experiment with Bacillus coagulans supernatant and cell wall fractions.
• Figure 33 is a schematic representation of how PMN migration begins in the blood stream and moves into the tissue via transwell migration plates.
• the present invention is directed to the discovery that non-pathogenic lactic acid- producing bacteria (i.e., "lactic acid bacteria"), such as Bacillus coagulans, are useful in boosting the immune system, i.e., increasing the level of activation of immune cells.
• Bacillus coagulans vegetative cells and/or spores are used or inactivated/dead Bacillus coagulans are used, e.g. , heat killed Bacillus coagulans.
• the administration of cell wall components or culture supernatants of Bacillus coagulans boosts the immune system by increasing cytokine production, activating the immune surveillance aspect of polymorphonuclear leukocytes (PMN), increasing immune cell chemotaxis, activating natural killer (NK) cells, and increasing monocyte phagocytosis.
• PMN polymorphonuclear leukocytes
• NK natural killer
• Bacterial species include Bacillus coagulans, e.g. , Bacillus coagulans hammer, preferably Bacillus coagulans hammer strain Accession No. ATCC 31284, or one or more strains derived from Bacillus coagulans hammer strain Accession No. ATCC 31284 (e.g., ATCC Numbers: GBI-20, ATCC Designation Number PTA-6085; GBI-30 or BC 30 , ATCC Designation Number PTA-6086; and GBI-40, ATCC Designation Number PTA-6087; see U.S. Patent No. 6,849,256 to Farmer).
• Bacillus coagulans e.g. , Bacillus coagulans hammer, preferably Bacillus coagulans hammer strain Accession No. ATCC 31284, or one or more strains derived from Bacillus coagulans hammer strain Accession No. ATCC 31284 (e.g., ATCC Numbers: GBI-20, ATCC Designation Number PTA-6085; G
• Bacillus coagulans vegetative cells or spores, Bacillus coagulans supernatant, and Bacillus coagulans cell wall components are useful in the present invention.
• Purified and/or isolated Bacillus coagulans vegetative cells or spores, Bacillus coagulans supernatant or Bacillus coagulans cell wall components are particularly useful as a probiotic in the compositions described herein.
• purified or substantially purified is meant Bacillus coagulans vegetative cells or spores, Bacillus coagulans supernatant, or Bacillus coagulans cell well components that are substantially free of contaminating microorganisms or other macromolecules, e.g., polysaccharides, nucleic acids, or proteins.
• a purified preparation contains at least 75%, 85%, 95% or about 100% of the desired composition and is substantially free of other sub-cellular components such as cytoplasmic organelles.
• a bacterial cell wall fraction is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or about 100% cell wall components.
• Such a preparation is sustainably free of cytoplasm intracellular organelles and secreted bacterial products.
• compositions include Bacillus coagulans vegetative cells or spores, Bacillus coagulans supernatant, or Bacillus coagulans cell wall components in the form of a powder, a dried cell mass, a stabilized paste, or a stabilized gel.
• Bacillus coagulans vegetative cells or spores, Bacillus coagulans cell wall and/or Bacillus coagulans culture supernatant is used in the methods described herein for boosting the immune system.
• the Bacillus coagulans cell wall and/or Bacillus coagulans culture supernatant is dried and reconstituted in water or other aqueous solution before use.
• Bacillus spores are heat and pressure-resistant and can be stored as a dry powder, they are particularly useful for formulation into and manufacture of products such as the various compositions described herein.
• a Bacillus species is well suited for the present invention, particularly species having the ability to form spores which are relatively resistant to heat and other conditions, making them ideal for storage (shelf- life) in product formulations.
• the compositions comprise Bacillus coagulans vegetative cells and/or spores.
• the cells/spores are formulated in a variety of compositions suited for use in an immune- boosting composition.
• the bacterium is present as a mixture of spores and vegetative cells.
• the bacterium is present as at least 90% spores, e.g., 95%, 98%, or 99% spores.
• the Bacillus coagulans cells are cultured in liquid in the absence of or with limited quantities of a food source to induce sporulation.
• heat gun spray drying kills about 50%, about 75%, about 90%, about 95%, or about 99% of vegetative cells prior to addition to the compositions of the invention.
• at least about 5%-25% of the bacteria in the composition are viable, e.g., at least about 25%-50%; at least about 50%-75%; or at least about 75%-99% of the bacteria are viable.
• the composition comprises at least about 1 x 10 6 to 1 x 10 7 ; at least about 1 x 10 7 to 1 x 10 8 ; or at least about 1 x 10 8 to 1 x 10 9 viable bacteria.
• the invention also provides Bacillus coagulans cell wall and/or Bacillus coagulans culture supernatant for use in the methods described herein for boosting the immune system.
• Bacillus coagulans bacteria, cell wall components, or culture supernatant in the form of a spray-dried powder is included in or on the surface of the composition described herein.
• the isolated Bacillus coagulans is in the form of a spore.
• the isolated Bacillus coagulans are at least 85%, at least 90%, at least 95%, or at least 99% pure spores.
• the isolated Bacillus coagulans is in the form of a vegetative cell.
• the isolated Bacillus coagulans are at least 85%, at least 90%, or at least 95% pure vegetative cells.
• the isolated Bacillus coagulans is in the form of a mixture of vegetative cells and spores.
• the Bacillus coagulans mixture is 90% spores, 10% vegetative cells; 75% spores, 25% vegetative cells; 60% spores, 40% vegetative cells; 50% spores, 50% vegetative cells; 60% vegetative cells, 40% spores; 75% vegetative cells; 25% spores; or 90% vegetative cells, 10% spores.
• the Bacillus and/or Bacillus coagulans isolated active agent e.g., Bacillus coagulans cell wall and/or Bacillus coagulans culture supernatant is applied using any of a variety of known methods including, for example, applying a powder, spray-drying the probiotic onto the composition, or soaking the composition in a solution containing the probiotic.
• the Bacillus coagulans cell wall and/or Bacillus coagulans culture supernatant is dried and reconstituted in water before use.
• Bacillus coagulans bacteria in the form of spray-dried powder administered directly.
• the composition comprises about 5 x 10 CFU Bacillus coagulans bacteria (per gram of composition) in the form of spray-dried powder.
• any of a variety of methods for placing the bacterial composition into a composition can be used.
• preferred methods include a "spray-dry" method in which the compositions are exposed in a low humidity chamber to an atomized mix containing a liquid composition, where the chamber is subsequently exposed to approximately 80-110 0 F to dry the liquid, thereby impregnating the material of composition with the components.
• a typical concentration is from approximately IxIO 7 to IxIO 12 CFU; IxIO 8 to IxIO 11 CFU; or 1x10 to 1x10 CFU of viable bacterium or spores/g of composition. Following drying, the composition is ready for immediate use or for storage in a sterile package.
• the active ingredients comprise between about 0.01% to about 10%; 0.01% to about 1%; or about 0.05% to about 0.1% by weight of the composition.
• the isolated Bacillus coagulans comprise about 1 mg to about 1O g; about 10 mg to about 1 g; or about 25 mg to about 75 mg by weight of the composition.
• the amount of Bacillus coagulans bacteria is about 5 x 10 7 colony forming units (CFU) of bacteria per gram of composition.
• the amount of bacteria is about 10 4 to 10 14 colony forming units (CFU) of bacteria per gram of probiotic composition (i.e., vegetative cells and/or bacterial spores), preferably 10 5 to 10 13 CFU/g of composition.
• the concentrations are 10 8 to 10 13 CFU/g; 10 9 to 10 12 CFU/g; or 10 10 to 10 11 CFU/g of composition.
• the amount of bacteria is about 1 x 10 6 CFU per gram of composition. The actual amount in a composition will vary depending upon the amounts of composition to be dispersed into the composition and upon routes of dispersal.
• the invention provides for storing the composition in a sterile package at room temperature prior to consumption. Alternatively, the composition is used immediately. In another aspect, the composition comprises at least 85%, at least 90%, at least 95% or 100% isolated Bacillus coagulans spores.
• Bacillus coagulans spores may be incorporated into any type of dry or lyophilized product which is dissolved or mixed with hot water, so long as the temperature of the Bacillus coagulans mixture is raised to the required heat-shock temperature (i.e., 80 0 C for 5 minutes) necessary for germination of the spores.
• the Bacillus coagulans spores may be incorporated into the dry or lyophilized product by the manufacturer.
• the Bacillus coagulans spores survive storage (shelf-life), i.e., retain viability or the ability to germinate at physiological conditions (e.g., ingestion), from about 12 days to about 2 years; from about 1 month to about 18 months; from about 3 months to about 1 year; or from about 6 months to about 9 months.
• Antimicrobial agents i.e., retain viability or the ability to germinate at physiological conditions (e.g., ingestion), from about 12 days to about 2 years; from about 1 month to about 18 months; from about 3 months to about 1 year; or from about 6 months to about 9 months.
• compositions of the invention also include known antimicrobial agents, known antiviral agents, known antifungal agents.
• the other agents in the compositions can be either synergists or active agents.
• the known antimicrobial, antiviral and/or antifungal agents are probiotic agents compatible with Bacillus coagulans .
• the compositions may also include known antioxidants, buffering agents, and other agents such as coloring agents, flavorings, vitamins or minerals. Thickening agents may be added to the compositions such as polyvinylpyrrolidone, polyethylene glycol or carboxymethylcellulose.
• the active agents are combined with a carrier that is physiologically compatible with the dermal or epithelial tissue of a human or animal to which it is administered. That is, the carrier is preferably substantially inactive except for surfactant properties used in making a suspension of the active ingredients.
• the compositions may include other physiologically active constituents that do not interfere with the efficacy of the active agents in the composition.
• a formulated composition of this invention may be completed in weight using any of a variety of carriers and/or binders.
• carriers are solid-based dry materials for formulations in tablet, granule or powdered form, and can be liquid or gel-based materials for formulations in liquid or gel forms.
• Typical carriers for dry formulations include trehalose, malto-dextrin, rice flour, micro-crystalline cellulose (MCC) magnesium stearate, inositol, FOS, gluco-oligosaccharides (GOS), dextrose, sucrose, and the like carriers.
• Other exemplary composition formulations include a pill, a capsule, or a suspension.
• Chemicals used in the present compositions can be obtained from a variety of commercial sources, including Spectrum Quality Products, Inc (Gardena, CA), Seltzer Chemicals, Inc., (Carlsbad, CA) and Jarchem Industries, Inc., (Newark, NJ).
• the Bacillus coagulans vegetative cells or spores, Bacillus coagulans supernatant, and Bacillus coagulans cell wall components of the invention are useful in enhancement of the immune response.
• Enhancement of the immune response comprises an increase in cytokine ⁇ e.g., IL-2, IL-4, IL-6, IL-IO, TNF- ⁇ , or IFN- ⁇ ) production or an increase in immune cell migration to an infection site.
• Immune enhancement also includes boosting the immune system by increasing cytokine production, activating the immune surveillance aspect of polymorphonuclear leukocytes (PMN), increasing immune cell chemotaxis, activating NK cells, and/or increasing monocyte phagocytosis.
• PMN polymorphonuclear leukocytes
• Bacillus coagulans Hammer bacteria (ATCC Accession No. 31284) was inoculated and grown to a cell density of about 10 8 to 10 9 cells/ml in nutrient broth containing 5 g Peptone, 3 g Meat extract, 10-30 mg MnS ⁇ 4 , and 1,000 ml distilled water, adjusted to pH 7.0, using a standard airlift fermentation vessel at 30 0 C.
• the range of MnSC> 4 acceptable for sporulation is 1 mg/1 to 1 g/1.
• the vegetative cells can actively reproduce up to 45°C, and the spores are stable up to 90 0 C.
• the B. coagulans bacterial cells or spores are collected using standard methods ⁇ e.g.
• the collected cells and spores can be lyophilized, spray-dried, air-dried or frozen.
• the supernatant from the cell culture is collected and used as source of extracellular agents secreted by B. coagulans.
• a typical yield from the above culture is in the range of about 10 9 to 10 10 viable spores and more typically about 100 to 150 billion cells/spores per gram before drying. Spores maintain at least 90% viability after drying when stored at room temperature for up to ten years, and thus the effective shelf life of a composition containing B. coagulans Hammer spores at room temperature is about 10 years.
• a culture of dried B. coagulans spores was prepared as follows. Ten million spores were inoculated into a one liter culture containing 24 g potato dextrose broth, 1O g of enzymic-digest of poultry and fish tissue, 5 g of FOS and 1O g MnS(V The culture was maintained for 72 hours under a high oxygen environment at 37°C to produce culture having about 150 billion cells per gram of culture. Thereafter, the culture was filtered to remove culture medium liquid, and the bacterial pellet was re-suspended in water and freeze-dried. The freeze-dried powder is then ground to a fine powder using standard good manufacturing practice (GMP).
• GMP standard good manufacturing practice
• Bacillus coagulans is a gram-positive rod that forms heat-resistant and acid- resistant spores. Oral consumption of spores or encapsulated spores allows transient colonization of the intestines with BC cultures. The spores germinate and the bacterial cultures grow and ferment the food in the intestinal lumen.
• Immune activation was induced in cultures of human immune cells when exposed to a) purified Bacillus coagulans culture supernatant (BCl), or b) purified Bacillus coagulans cell wall components (BC2). These two fractions were utilized to characterize the interactions between Bacillus coagulans and immune cells in vivo, e.g., the Lamina Propria or Peyer's Patches located in the lumen of the intestines. Bacillus coagulans supernatant (BCl) and Bacillus coagulans cell wall components (BC2) were tested in parallel using a panel of cell- based assays in vitro.
• BCl Bacillus coagulans culture supernatant
• BC2 Bacillus coagulans cell wall components
• BC cell walls contain unique components that interact with immune cells in such a way as to activate or boost the immune system. BC also secretes metabolites and/or other factors that are produced when BC is growing in the environment of the small intestine. Such metabolites/factors include, but are not limited to antioxidant and antiinflammatory compounds.
• a sample of Bacillus coagulans spores was heat-activated at 50 0 C and inoculated in liquid medium. The sample was incubated at 37°C for 24 hours. This time period allows the formation of a log-phase bacterial culture where death and bacterial breakdown is not prominent. After the incubation, the two fractions ⁇ Bacillus coagulans supernatant (BCl) and Bacillus coagulans cell wall components (BC2)) were prepared. The initial separation occurred by decanting the entire culture into a 5OmL vial followed by centrifugation at 2400 rpm. This resulted in the bacteria forming a pellet. The supernatant was gently decanted into a new vial.
• BCl Bacillus coagulans supernatant
• BC2 Bacillus coagulans cell wall components
• the thawed slush was transferred to an Eppendorf vial and washed twice in physiological saline using high speed centrifugation. Then the pellet was transferred to a glass vial and subjected to bead milling using low-protein-binding Zirconium beads with a diameter of 200 micrometer. The milling was performed by repeated 'pulsing' using a Vortex mixer. This method is effective to break up cell walls. The beads were removed and the slush containing the broken cell wall fragments were sterile-filtered into multiple aliquots that were frozen immediately and stored at -20 0 C. For later assays, one aliquot was thawed on each testing day. Purification of peripheral blood mononuclear cells and polymorphonuclear cells
• phagocytic activity was performed using human PMN cells.
• the choice of particles for phagocytosis was carboxylated Fluorospheres (Molecular Probes, Eugene OR).
• An aliquot of 0.05 mL Fluorobeads was removed from the stock bottle into a 1.5 mL microcentrifuge tube and washed twice in PBS. Fluorobeads were then re-suspended in 7.5 mL RPMI 1640.
• PMN cells were plated into 96-well plates in RPMI- 1640 at a concentration of 2 x 10 cells/mL.
• the PMN cell is a highly active and migratory cell type (Figure 3).
• the differential effect on PMN cell migration towards the bacterial peptide formyl-Met-Leu-Phe (f-MLP) and two different inflammatory chemo-attractants IL-8 and Leukotriene B4 (LTB4) was tested.
• the following experimental model was performed in quadruplicate in order to obtain data significance. Cells were incubated with 10-fold serial dilutions of GBI-30 (GanedenBC ) supernatant or cell wall fractions for 10 minutes in a polystyrene round-bottom tube before plating commenced.
• the Millipore trans- well (3.0 ⁇ m pore size) migration plate was coated with 50 ⁇ g/mL Fibronectin for a period of 30 minutes.
• Chemoattractants and RPMI 1640 were then added to the appropriate bottom chamber wells of the trans-well migration plate in a volume of 150 ⁇ L: f-MLP (10 nM), Interleukin-8 (10 ⁇ g/mL), and Leukotriene B4 (10 nM). Fibronectin was removed from the top wells by aspiration before plating of cells.
• PBMC peripheral blood mononuclear cells
• the two cell types were loosely pelleted by a brief 30-second centrifugation at 2400 rpm followed by incubation at 37 0 C for 45 minutes. Cells were transferred to V-bottom microtiter plates for processing and staining. Cells were stained with CD3-PerCP, CD56-PE and CD107a-FITC. The expression of CD 107a on the NK cells was determined by flow cytometry. The CD3 negative, CD56 positive NK cells were differentiated from the K562 cells based on forward and side scatter properties, and from other lymphocytes by electronic gating on CD3 " , CD56 + cells, followed by evaluation of fluorescence intensity for CD 107a. Induction of natural killer cell activation markers and immuno-staining
• PBMC freshly isolated PBMC were plated in a sterile U-bottom 96-well culture plates (NUNC, Denmark) and treated with serial dilutions of test products. For activation of natural killer (NK) and natural killer T (NKT) cells the incubation time was 18 hours. Cells were transferred to V- bottom 96-well plates (NUNC Denmark) and washed in IF buffer (PBS containing 1% bovine serum albumin and 0.02% sodium azide).
• IF buffer PBS containing 1% bovine serum albumin and 0.02% sodium azide
• Cells were re-suspended in 0.05 mL IF buffer and monoclonal antibodies were added in previously established optimal quantities (CD3-PerCP, CD56-PE, CD69-FITC, and CD25-FITC: 8 ⁇ L/sample), and incubated in the dark at room temperature for 10 minutes. The cells were washed twice with an additional 0.15 mL of PBS with 0.02% azide. Following centrifugation and aspiration of the supernatant, the cells were re- suspended in 0.05 mL PBS with 0.02% azide and transferred to 5 mL polystyrene round-bottom tubes each containing 0.4 mL of 1% formalin.
• Mitogens were added at a concentration of 5 ⁇ L of PWM (200 ⁇ g/mL) and 4 ⁇ L of PHA (2 ⁇ g/mL) to initiate proliferation.
• the plate was sealed with parafilm and was incubated at 37 0 C, 5% CO 2 for 5 days. After 5 days the cells were transferred to a flat-bottom black 96-well plate and the relative cell numbers in each culture well quantified by CyQuant® staining and a Tecan Spectrafluor fluorescence plate reader.
• PMN polymorphonuclear — white blood cell
• BC2 Bacillus coagulans cell wall components
• NK cells are important in the defense against cancer cells and viruses. Both Bacillus coagulans supernatant (BCl) and Bacillus coagulans cell wall components (BC2) activated Natural Killer (NK) cells. Both fractions enhanced the aggressive secretion of killer substances from NK cells when the NK cells subsequently were contacted with tumor cells. Anti-inflammatory effects
• Bacillus coagulans GBI-30 (GanedenBC30TM) fractions were introduced to PMN cells at very low doses. The PMN cells were then coached to migrate towards an inflammatory mediator, Leukotriene B4. This assay mimics the PMN cell's role in maintaining an inflammatory cascade.
• Ganeden BC30TM supports or inhibits the migration of the inflammatory PMN cells, depending on whether the PMN cell is engaged in normal immune surveillance or is engaged in an inflammatory response.
• the dilutions on the X-axis refers to the tested dilution of each Bacillus coagulans (BC) fraction.
• a 1: 100 dilution is 100- fold dilution of the initial frozen stock solution.
• ROS reactive oxygen species
• PMN Human polymorph nucleated cells
• BCl Bacillus coagulans supernatant
• BC2 Bacillus coagulans cell wall components
• the cells were washed and loaded with the DCF-DA dye, which fluoresces upon exposure to reactive oxygen species. Oxidation was triggered by addition Of H 2 O 2 .
• the fluorescence intensity of the PMN cells was evaluated by flow cytometry. The low fluorescence intensity of untreated control cells served as a baseline, while PMN cells treated with H 2 O 2 alone served as a positive control.
• test product If the fluorescence intensity of PMN cells exposed to Bacillus coagulans, and subsequently exposed to H 2 O 2 is reduced compared to exposure to H 2 O 2 alone, the test product has anti-inflammatory effects. By contrast, if the fluorescence intensity of PMN cells exposed to a test product is increased compared to H 2 O 2 alone, a test product has pro-inflammatory effects.
• the PMN cell is capable of signaling by both anti- and proinflammatory mechanisms, which can lead to either enhancement or reduction of the production of reactive oxygen species (ROS).
• ROS reactive oxygen species
• Both BCl and BC2 showed a clear inhibition of the spontaneous formation of reactive oxygen species in PMN cells.
• the effect of BC2 showed a dose-dependent inhibition of ROS formation, whereas the effect of BCl showed stronger antiinflammatory effect at the lowest doses tested.
• BCl and BC2 presented roughly a 25% inhibition of baseline ROS formation at the highest dilution of 1 :1000.
• the presence of BCl (1 : 1000) reduced spontaneous ROS formation by 22% (i > ⁇ 0.003).
• BC2 (1 : 1000) showed a similar effect on lowering ROS formation (P ⁇ 0.004).
• the PMN cell is a highly active and migratory cell type that plays a major role in immune surveillance.
• the migratory behavior of PMN is divided into at least two types: a) random migration and b) directed migration. Random migration is part of normal immune surveillance, while directed migration is migration toward specific chemoattractants.
• test products may specifically reduce directed PMN migration towards the inflammatory mediators IL-8 and/or LTB4, while allowing PMN migration toward bacterial peptides as part of the normal anti-bacterial immune defense.
• the testing of migration towards several inflammatory chemo-attractants helps identify selective responses in this in vitro system, which closely mimics the rat paw edema in vivo model of inflammation.
• the assay allows for the differentiation between antibacterial immune defense mechanisms and inflammation response mechanisms.
• Freshly purified PMN cells were cultured in double- chamber migration plates, the bottom chamber mimicking tissue, and the top chamber mimicking the blood stream (Figure 3). Cells were plated in the top chambers with or without test products, while different chemoattractants were placed in the bottom chambers. For control wells, cells were placed in the top chamber without a test product and chemo-attractant was not placed in the bottom wells. In this manner, evaluation of baseline random migration was determined. All assays were performed in triplicate, and repeated at least 3 times with consistent results using freshly isolated cells from three different healthy human donors.
• Bacillus coagulans supernatant (BCl) was tested at one extra dilution, compared to Bacillus coagulans cell wall components (BC2). Both fractions of the GanedenBC 30TM (Bacillus coagulans) induced the random migration of PMN cells, indicating that both Bacillus coagulans supernatant (BCl) and Bacillus coagulans cell wall components (BC2) activated the immune surveillance aspect of PMN cells ( Figure 4). BCl (1 :10) increased the random migration by 300% (p ⁇ 0.001), and BC2 (1: 10) increased the random migration by 200% (p ⁇ 0.005).
• BCl and BC2 decreased the amount of f-MLP directed migration, indicating that different levels of Bacillus coagulans have different effects on immune cells in the gut.
• BCl (1 :10,000) decreased f-MLP directed migration by 11%; however, this was not statistically significant, and BC (1 :1000) decreased f-MLP directed migration by 46% (P ⁇ 0.005).
• both BCl and BC2 fractions also had a dual effect on LTB4 directed PMN migration.
• both BCl and BC2 demonstrated a dose-dependent reduction in PMN migration towards LTB4 (Figure 7).
• the 1 : 1000 dilution of BC2 inhibited migration by 52% (p ⁇ 0.002).
• Cells treated with 1 :1000 (p ⁇ .008) and 1: 10000 (pO.002) dilutions of BCl also showed antiinflammatory effect that were highly statistically significant.
• the 1 :10 dilution of BCl resulted in a significant increase in PMN migration towards LTB4 (p ⁇ 0.003). Chemoattractant effect on PMN cell migration
• the BC fractions were added to the bottom chambers and direct chemoattractant properties of the BC fractions were measured.
• the BC fractions were placed in the bottom chambers in combination with each of the following chemoattractants: bacterial f-MLP, IL-8 and Leukotriene B4 in order to examine the synergistic affects on PMN migration in the presence of both the BC fractions and a known chemoattractant.
• the two Bacillus coagulans fractions also had very different effects on the IL-8 directed migration. All doses of BCl enhanced the IL-8 directed migratory activity. The chemoattractant effect of BCl (1: 10) was statistically significant (P ⁇ 0.00001). BC2 had a dual effect. At higher doses BC2 enhanced IL-8 induced migration. However, at the lowest dose tested, this fraction reduced migration towards IL-8. The interaction of PMN cells with BC2 made the cells much less responsive to IL-8. BC2 (1 : 1000) treated cells inhibited migration by 49% (P ⁇ 0.004).
• BC2 had a dual effect. At the higher dose, it enhanced LTB4 induced migration. However, at the 1 : 100 dose, this fraction reduced migration towards LTB4. The interaction of PMN cells with BC2 made the cells less responsive to LTB4. BC2 (1 : 100) treated cells inhibited migration by 11% (P ⁇ 0.01).
• Phagocytosis of microbial particles is an important part of the innate immune response. It is a rapid process, and the effect of a test product on enhancing this cellular function can be almost immediate. Phagocytosis was measured by how well PMN cells engulfed green fluorescent carboxylated fluoro spheres. The mean fluorescence intensity (MFI) of phagocytic cells was then evaluated by flow cytometry. Freshly purified peripheral blood mononuclear cells were pretreated with either Bacillus coagulans supernatant (BCl) or Bacillus coagulans cell wall components (BC2) for 3 minutes, and then introduced to fluorescent micro-particles mimicking bacteria.
• BCl Bacillus coagulans supernatant
• BC2 Bacillus coagulans cell wall components
• the cells were allowed to ingest particles for 2 minutes, after which free micro- particles were removed by centrifugation.
• the fluorescence intensity of the phagocytes was then evaluated by flow cytometry. Electronic gating was performed on the monocyte population, and the analysis was performed by measuring the mean fluorescence intensity (MFI FL3). A faster or stronger rate of phagocytosis results in a higher number of fluorescent micro-particles per cell.
• BCl at the dilution of 1 : 10 increased monocyte phagocytosis. This increase of 28% was highly significant (P ⁇ 0.01).
• BCl and BC2 increased phagocytosis in PMN cells compared to the negative controls. Exposure of PMN cells to BCl at the 1:10 dilution increased phagocytosis by 40% (/> ⁇ 0.02). Exposure of PMN cells to BC2 at the 1 :10 dilution increased phagocytosis by 25% (/> ⁇ 0.008). Further dilutions of both products resulted in reduced PMN phagocytosis (p ⁇ 0.05).
• Figures 30A and 30B represent photographs taken from a fluorescence microscope.
• the control shows an untreated PMN cell that has engaged in phagocytosis (Figure 30A).
• the green beads are carboxylated fluorospheres that mimic bacterial particles.
• Figure 30B shows a PMN cell that has been treated with BC2. This cell has ingested many more fluorospheres than the untreated cell in Figure 30A.
• This figure in combination with the data above, indicates that Bacillus coagulans increases the capacity of phagocytes for engulfing foreign material.
• Natural Killer (NK) cells are involved in the primary defense mechanisms against transformed cells and viruses. These cells travel in the blood stream in a state of rest, but can be immediately activated to a) kill cancer cells by either cell contact or secretion of cytotoxic compounds such as perforin and granzyme, b) proliferate, and c) secrete substances that attract other cells into the site.
• cytotoxic compounds such as perforin and granzyme
• BCl and BC2 the changes in expression of the NK activation cell surface marker CD69 were examined. The increased expression of this marker has been associated with an increased cytotoxic activity of NK cells (Clausen et al, 2003 Immunobiol, 207(2):85-93).
• Freshly purified human peripheral blood mononuclear cells were used for these assays.
• the cells were plated in 96-well micro-assay plates in triplicate. Negative control wells in triplicate were left untreated. Positive controls were treated with IL-2 at a dose of 100 international units per mL (IU/mL).
• IU/mL international units per mL
• Figure 14 depicts the change in mean fluorescence intensity of the NK activation marker CD69, following exposure of NK cells to either Bacillus coagulans supernatant (BCl) or Bacillus coagulans cell wall components (BC2). Both BCl and BC2 showed a clear dose- dependent induction of the expression of CD69 on NK cells. The effect reached high statistical significance for both BCl and BC2 at a 1 :400 dilution, where CD69 expression was increased by 32% (p ⁇ 0.0l) for BCl, and by 36% for BC2 (p ⁇ 0.003).
• BCl Bacillus coagulans supernatant
• BC2 Bacillus coagulans cell wall components
• Figure 15 shows the expression of CD25 on NKT cells. For both BCl and BC2, there was not a large difference in CD25 expression compared to baseline levels. While the 1 : 100 dilution of BCl resulted in an increase in expression, this change did not reach statistical significance. No changes in CD25 expression on T cells were observed when comparing untreated cells to those treated with either BCl or BC2. Externalization of CD 107a on NK cells in response to tumor cells
• NK cells One of the functions of NK cells is to kill tumor cells and virus-infected cells via cell-cell contact and by secretion of substances such as Perform.
• the CD 107a receptor expressed on the interior of granules in the cytoplasm of NK cells is transiently brought to the cell surface.
• CD 107a expression on NK cells is a measure of their cytotoxic activity by secretion of cytotoxic substances.
• Freshly-purified human peripheral blood mononuclear cells were used for this assay.
• the cells were plated in round-bottom 96-well micro-assay plates, and treated with serial dilutions of the test products in triplicate. Negative control wells in triplicate were left untreated. All other wells were used for addition of the NK-cell sensitive K562 tumor cell line, widely used in NK cell cytotoxicity assays. Positive control wells were left without adding test products. All remaining wells were treated with serial dilutions of test products. The two cell types were brought physically together by a brief 15-seconds centrifugation, and incubated for 45 minutes at 37 0 C. Cells were transferred to V-bottom microtiter plates for processing and staining.
• CD 107a on the NK cells was analyzed by flow cytometry, where the NK cells were differentiated from the other lymphocytes based on positive staining for CD3 and CD56, and from the K562 cells based on size. Cytotoxic activity of NK cells in vitro was measured. The response in this assay predicts a similar response to non-malignant, virally infected cells.
• Figure 16 shows the change in mean fluorescence intensity (MFI) of CD 107a expression on natural killer cells that have been exposed to tumor cells, with or without the addition of BCl or BC2.
• MFI mean fluorescence intensity
• Example 6 Support of adaptive immune function: Modulation of lymphocyte proliferation and cytokine production in response to two known mitogens.
• test products were examined for mitogenic potential, that is, whether they induce cell division in healthy human lymphocytes. Simultaneous to the test of mitogenic potential, the test products were examined for their effect on cells responsible for the adaptive immune defense, that is, T and B lymphocytes.
• the lymphocyte proliferation assay offers a simple method to assess whether the compositions alter lymphocyte responsiveness to known signals. A change in the proliferative response to known mitogens in the presence of a composition indicates an immunomodulatory effect, such as T and B lymphocyte signaling and activation.
• compositions were tested in serial dilutions in the presence and absence of mitogens.
• Two mitogens were tested in parallel: Phytohemagglutinin (PHA), which is a T cell mitogen that will induce T cell proliferation, and Pokeweed Mitogen (PWM), which is a mitogen that requires the collaboration of T cells, B cells and monocytes in the culture.
• PHA Phytohemagglutinin
• PWM Pokeweed Mitogen
• Freshly purified human peripheral blood mononuclear cells (PBMC) were cultured in the absence versus presence of serial dilutions of the compositions. Three parallel sets of cultures were established, where one tested the direct effect of test product on lymphocyte proliferation, and the two others examined the interference of the composition with response to the known mitogens. Positive controls included cells treated only with a mitogen in the absence of test product. A change (increase, decrease) of mitogen-induced proliferation is a strong indication of the presence of immuno modulating compounds.
• BCl nor BC2 had a mitogenic effect on lymphocyte proliferation following five days incubation at 37 0 C with product and culture media.
• BCl and BC2 showed a reduction in lymphocyte proliferation in the presence of PHA and PWM ( Figures 17 and 18). This reduction was significant at all doses of BCl in the presence of both PHA and PWM (p ⁇ 0.02) and was significant for the two highest concentrations of BC2 (p ⁇ 0.02). Additionally, high statistical significance was reached for BCl at the 1: 10 (p ⁇ 0.003) and 1: 100 (p ⁇ 0.002) doses and BC2 at the 1 :10 (p ⁇ 0.004) dose in the presence of PHA and BCl at the 1 : 100 (p ⁇ 0.005) dose and BC2 at the 1 : 10 (p ⁇ 0.002) and 1 : 100 (p ⁇ 0.006) doses in the presence of PWM. Cytometric Bead Array
• a flow cytometry-based Thl/Th2 cytokine bead array (CBA) for the 6 cytokines IL-2, IL-4, IL-6, IL-10, TNF- ⁇ and INF- ⁇ was used to evaluate the levels of cytokines present in the supernatants from 5-day lymphocyte cultures.
• CBA flow cytometry-based Thl/Th2 cytokine bead array
• Lymphocytes were also cultured (with or without product) in the presence of 2 different mitogens.
• Phytohemagglutinin (PHA) was used to induce T cell proliferation
• PWM Pokeweed Mitogen
• Comparisons were made between lymphocytes cultured in the presence of no product and lymphocytes cultured in the presence of 1:100 dilutions of either BCl or BC2. This data is presented in separate graphs for each individual cytokine and also compares changes in cytokine levels in lymphocytes that were cultured without mitogens either without product or with the 1 : 100 dilutions of BC 1 and BC2.
• TNF- ⁇ production was slightly lower than untreated PBMC in the presence of both BCl and BC2 ( Figure 25A and 25B). This mild reduction was not statistically significant for either BCl or BC2.
• Treatment of PBMC with either BCl or BC2 in the presence of PHA resulted in 2-fold decreases in TNF- ⁇ expression that were statistically significant for both BCl (P ⁇ 0.002) and BC2 CP ⁇ 0.006).
• treatment of PBMC with BCl and BC2 in the presence of PWM resulted in strong increases in TNF- ⁇ levels.
• BCl treatment produced an 11 -fold increase (/0.003) and BC2 treatment a 22-fold increase (PO.001).
• the culture supernatant and cell wall fractions were tested in the Cell-based Antioxidant Protection in Erythrocytes (CAP-e) assay, a bioassay for antioxidants test ( Figures 27-29).
• This assay allows assessment of antioxidant potential in a method that is comparable to the oxygen radical absorbance capacity (ORAC) test, but only allows measurement of anti-oxidants that are able to cross the lipid bilayer cell membrane.
• ORAC oxygen radical absorbance capacity
• red blood cells RBC
• This assay is particularly useful to assess antioxidants from complex natural products in a cell-based system.
• Freshly purified human RBC were washed repeatedly in physiological saline, and then exposed to the test compositions. During the incubation with a test product, any antioxidant compounds able to cross the cell membrane can enter the interior of the RBC. RBC were then washed to remove compounds that were not absorbed by the cells, and loaded with the DCF-DA dye, which turns fluorescent upon exposure to reactive oxygen species. Oxidation was triggered by addition of the peroxyl free radical generator AAPH. The fluorescence intensity was evaluated. The low fluorescence intensity of untreated control cells serve as a baseline, and RBC treated with AAPH alone serve as a positive control for maximum oxidative damage. An observation of a reduced fluorescence intensity of RBC exposed to a test product and subsequently exposed to AAPH, indicates that the test product contains antioxidants available to penetrate into the cells and protect these from oxidative damage.
• BC-I cell-free BC supernatants
• BC-2 cell-free cell wall fractions
• both BCl and BC2 inhibited spontaneous ROS formation and reduced ROS formation when oxidative stress had been applied to PMN's.
• both BCl and BC2 increased the migration of PMN cells towards a bacterial peptide, indicating an enhancement of the immune surveillance function of PMN in detecting bacteria.
• both BCl and BC2 decreased the migration of PMN cells towards a bacterial peptide, indicating that at low concentrations, the compounds present in the supernatant and cell wall preparations of Bacillus coagulans had an immunomodulatory effect on the ability of the PMN to respond to the bacterial peptide signal. This effect underlies processes that dictate how the immune system responds to a bacterial infection vs.
• BCl resident (beneficial) commensal bacteria in the gut.
• the highest dose of BCl increased monocyte phagocytosis.
• BC2 did not increase monocyte phagocytosis.
• BCl and BC2 at the strongest dose increased PMN phagocytosis.
• NK expression (CD69) was increased in all dilutions of BCl and BC2 tested.
• BCl and BC2 both deceased lymphocyte proliferation at all doses.
• Both Bacillus coagulans fractions reduced IL-2 and TNF.
• These cytokines are known THl cytokines which is directed at macrophage activation. However, a mild increase was noticed for IFN- ⁇ production for both Bacillus coagulans fractions.
• a sample of BC spores was heat-activated and inoculated in a liquid culture medium. Sample is incubated at 37°C for 24 hours. This time period allows the formation of a log-phase bacterial culture where death and bacterial breakdown is not prominent. After the incubation, the two fractions (cell wall and supernatant) are prepared. The initial separation is accomplished by decanting the entire culture into a 5OmL vial and centrifugation at 2400 rpm. Bacterial aggregates form a pellet. The supernatant is gently be decanted into a new vial.
• the pellet is transferred to a glass vial and subjected to bead milling using low-protein-binding Zirconium beads with a diameter of 100 micrometer.
• the milling is performed by repeated 'pulsing' using a Vortex mixer.
• the beads are removed and the slush containing the broken cell wall fragments are sterile-filtered into multiple aliquots that will be frozen immediately at -8O 0 C.
• Electrophoresis is used to understand the protein and polysaccharide makeup of Bacillus coagulans fractions and spores.
• a typical protein gel electrophoresis method is shown in Figure 31. This process separates the proteins and polysaccharides by molecular weight and gives a valuable fingerprint for each of the BC fractions. Electrophoretic separation provides information about the relative quantity of specific proteins and polysaccaharides in the product.
• the PMN cell is a highly active and migratory cell type. Bacillus coagulans fractions have strong anti- inflammatory effects when exposed to the known inflammatory cytokine LTB4. Crude BC cell wall and BC supernatant are fractionated into the following molecular weight ranges: a) ⁇ 3kDa, b) 3-30 kDa, and c) 30-20OkDa. Similar volumes of Bacillus coagulans cell wall and supernatant are placed into centrifugation columns that filter out specific molecular weight fractions. After centrifugation the remaining volumes are serial diluted and placed in with the PMN' s before plating into the top chamber.
• Freshly purified PMN cells cultures are set up in double-chamber migration plates, where the bottom chamber mimics tissue, and the top chamber mimics the blood stream as described in Figure 33. Cells are plated in the top chambers with and without test products, and the different chemo-attractant (LTB4) is present in the bottom chambers. All assays are performed in triplicates, and repeated at least 3 times with consistent results. The testing of migration towards the inflammatory chemo-attractant LTB4 identifies selective responses in this in vitro system, which closely mimics some in vivo models of inflammation, such as rat paw edema. The assay allows a distinction between normal PMN defense mechanisms versus response to inflammation.
• the anti-inflammatory activity of Bacillus coagulans spores is also examined. These assays identify which molecular weight compounds are responsible for the anti-inflammatory effects of BC supernatant and cell wall fractions.
• Natural Killer Cell Activation (CD69 expression) Crude BC cell wall and BC supernatant are fractionated into the following molecular weight ranges: a) OkDa, b) 3-30 kDa, and c) 30-20OkDa. As described above, both BC fractions activated NK cells. Induction of the CD69 activation marker on the NK cells is determined. Freshly purified human peripheral blood mononuclear cells are used for these assays. The cells are plated in 96-well micro-assay plates in triplicate.
• Negative control wells in triplicate are left untreated. Positive controls are treated with IL-2 at a dose of 100 international units per mL (IU/mL). After 18 hours of culture, cells are stained for the activation molecule CD69 on the surface of CD3-negative, CD56-positive NK cells.
• Bioactivity of Bacillus coagulans supernatant and cell wall components is also assessed after drying and reconstitution to determine if bioactivity is preserved after drying.
• This assay identifies which molecular weight compounds are responsible for the NK cell activating effects of BC supernatant and cell wall fractions. The ability of Bacillus coagulans spores to activate NK cells is also examined. Cytokine production
• Example 10 A controlled trial to evaluate the effects of GB 1-30 (GanedenBC ) (viable cells and spores) on the immune system
• the beneficial effect of GanedenBC ⁇ Bacillus coagulans GBI-30, ATCC Designation Number PTA-6086) on the immune system in healthy individuals was evaluated when challenged with the adenovirus and influenza. Studies were also carried out to determine the beneficial effect of GanedenBC ⁇ Bacillus coagulans GBI-30, ATCC Designation Number PTA-6086) in health individuals on %CD3CD69 cells- a marker for T-lymphocyte activity. Ten healthy adult subjects were recruited for this study. No concurrent illness or recent immunization was allowed. Blood was drawn at baseline at day O. Subjects were instructed to consume 1 capsule daily containing 500 million CFU's of GanedenBC daily for 30 days. Blood was drawn again on day 30. Due to one subject being statistically different at baseline, only 9 subjects were used in the final analysis.
• Blood samples were taken and stimulated with either the adenovirus or influenza A and incubated for 24 hours and then vortexed. 100 microliters of the sample was drawn off and of that 20 microliters were used for the %CD3CD69 testing. 900 microliters from the remaining samples were drawn and centrifuged and the plasma removed. Samples of the plasma were taken and used in the cytokine testing. Various cytokines were tested, and ones with statistically changes are noted below.

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• 2010
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